GABAA receptor-mediated inhibition depends on the maintenance of low level intracellular [Cl−] concentration, which in adult depends on neuron specific K+-Cl− cotransporter-2 (KCC2). Previous studies have shown that KCC2 was downregulated in both epileptic patients and various epileptic animal models. However, the temporal relationship between KCC2 downregulation and seizure induction is unclear yet. In this study, we explored the temporal relationship and the influence of KCC2 downregulation on seizure induction. Significant downregulation of plasma membrane KCC2 was directly associated with severe (Racine Score III and above) behavioral seizures in vivo, and occurred before epileptiform bursting activities in vitro induced by convulsant. Overexpression of KCC2 using KCC2 plasmid effectively enhanced resistance to convulsant-induced epileptiform bursting activities in vitro. Furthermore, suppression of membrane KCC2 expression, using shRNAKCC2 plasmid in vitro and shRNAKCC2 containing lentivirus in vivo, induced spontaneous epileptiform bursting activities in vitro and Racine III seizure behaviors accompanied by epileptic EEG in vivo. Our findings novelly demonstrated that altered expression of KCC2 is not the consequence of seizure occurrence but likely is the contributing factor.

Phenol red is widely used in cell culture as a pH indicator. Recently, it also has been reported to have estrogen-like bioactivity and be capable of promoting cell proliferation in different cell lines. However, the effect of phenol red on primary neuronal culture has never been investigated. By using patch clamp technique, we demonstrated that hippocampal pyramidal neurons cultured in neurobasal medium containing no phenol red had large depolarization-associated epileptiform bursting activities, which were rarely seen in neurons cultured in phenol red-containing medium. Further experiment data indicate that the suppressive effect of the phenol red on the abnormal epileptiform burst neuronal activities was U-shape dose related, with the most effective concentration at 28 µM. In addition, this concentration related inhibitory effect of phenol red on the epileptiform neuronal discharges was mimicked by 17-β-estradiol, an estrogen receptor agonist, and inhibited by ICI-182,780, an estrogen receptor antagonist. Our results suggest that estrogen receptor activation by phenol red in the culture medium prevents formation of abnormal, epileptiform burst activity. These studies highlight the importance of phenol red as estrogen receptor stimulator and cautions of careful use of phenol red in cell culture media.

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We have previously reported that cyclothiazide (CTZ) evokes epileptiform activities in hippocampal neurons and induces seizure behavior. Here we further studied in vivo the sensitivity of the hippocampal CA1 neurons in response to CTZ in epileptogenesis in comparison with two other classic convulsants of kainic acid (KA) and pentylenetetrazol (PTZ). CTZ administered intracerebral ventricle (i.c.v.) induced epileptiform activities from an initial of multiple evoked population spikes, progressed to spontaneous spikes and finally to highly synchronized burst activities in hippocampal CA1 neurons. PTZ, when given by subcutaneously, but not by intracerebral ventricle injection, evoked similar progressive epileptiform activities. In contrast, KA given by i.c.v. induced a quick development of epileptiform burst activities and then shortly switched to continuous high frequency firing as acute status epilepticus (ASE). Pharmacologically, alprazolam, a high-potency benzodiazepine ligand, inhibited CTZ and PTZ, but not KA, induced epileptiform burst activities while GYKI 53784, an AMPA receptor antagonist, suppressed CTZ and KA but not PTZ evoked epileptiform activities. In conclusion, CTZ and PTZ induced epileptiform activities are most likely to share a similar progressive pattern in hippocampus with GABAergic mechanism dominant in epileptogenesis, while CTZ model involves additional glutamate receptor activation. KA induced seizure in hippocampus is different to that of both CTA and PTZ. The results from this study indicate that hippocampal neurons respond to various convulsant stimulation differently which may reflect the complicated causes of the seizure in clinics.

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BackgroundThe previous studies have demonstrated the reduction of thiamine diphosphate is specific to Alzheimer’s disease (AD) and causal factor of brain glucose hypometabolism, which is considered as a neurodegenerative index of AD and closely correlates with the degree of cognitive impairment. The reduction of thiamine diphosphate may contribute to the dysfunction of synapses and neural circuits, finally leading to cognitive decline.ResultsTo demonstrate this hypothesis, we established abnormalities in the glucose metabolism utilizing thiamine deficiency in vitro and in vivo, and we found dramatically reduced dendrite spine density. We further detected lowered excitatory neurotransmission and impaired hippocampal long-term potentiation, which are induced by TPK RNAi in vitro. Importantly, via treatment with benfotiamine, Aβ induced spines density decrease was considerably ameliorated.ConclusionsThese results revealed that thiamine deficiency contributed to synaptic dysfunction which strongly related to AD pathogenesis. Our results provide new insights into pathogenesis of synaptic and neuronal dysfunction in AD.

Potassium chloride co-transporter 2 (KCC2), a major chloride transporter that maintains GABAA receptor inhibition in mature mammalian neurons, is down-regulated in the hippocampus during epileptogenesis. Impaired KCC2 function accelerates or facilitates seizure onset. Calpain, with two main subtypes of m- and μ-calpain, is a Ca2+-dependent cysteine protease that mediates the nonlysosomal degradation of KCC2. Although recent studies have demonstrated that calpain inhibitors exert antiepileptic and neuroprotective effects in animal models of acute and chronic epilepsy, whether calpain activation affects seizure induction through KCC2 degradation remains unknown. Our results showed that: (1) Blockade of calpain by non-selective calpain inhibitor MDL-28170 prevented convulsant stimulation induced KCC2 downregulation, and reduced the incidence and the severity of pentylenetetrazole (PTZ) induced seizures. (2) m-calpain, but not μ-calpain, inhibitor mimicked MDL-28170 effect on preventing KCC2 downregulation. (3) Phosphorylation of m-calpain has been significantly enhanced during seizure onset, which was partly mediated by the calcium independent MAPK/ERK signaling pathway activation. (4) MAPK/ERK signaling blockade also had similar effect as total calpain blockade on both KCC2 downregulation and animal seizure induction. The results indicate that upregulated m-calpain activation by MAPK/ERK during convulsant stimulation down regulates both cytoplasm- and membrane KCC2, and in turn facilitates seizure induction. This finding may provide a foundation for the development of highly effective antiepileptic drugs targeting of m-calpain.